Herpes simplex virus 1 (HSV-1) encephalitis (HSE) is the most common form of sporadic viral encephalitis in Western countries. We discovered that childhood HSE may result from inborn errors of Toll-like receptor 3 (TLR3)-dependent, interferon (IFN)-???-mediated immunity against primary infection by HSV-1 (mutations in dsRNA-sensing endosomal receptor TLR3 and TLR3-pathway molecules UNC-93B, TRIF, TRAF3 and TBK1). Rare children with HSE and other infections carry mutations that impair immunity more broadly (STAT1 or NEMO). Thanks to NIH R01NS072381, we showed that forebrain neurons and pro-oligodendrocytes, derived from TLR3- and UNC93B-deficient induced pluripotent stem cells (iPSCs), (i) have markedly abnormal cellular responses to poly(I:C), a TLR3 mimic of dsRNA, unlike the leukocyte subsets tested, and (ii) are highly susceptible to HSV-1 infection, unlike other central nervous system (CNS)-resident cells tested. These data suggested that childhood HSE results from inborn errors of non-hematopoietic, CNS-specific, intrinsic immunity, affecting neurons and oligodendrocytes in particular. The goal of this renewal application is to dissect in greater depth, and from three complementary angles, the neuron-intrinsic pathogenesis of HSE. First, we will devise a faster protocol to differentiate and test iPSC-derived forebrain neurons from healthy controls and four groups of patients, mutated in (i) HSE-causing TLR3-pathway genes (TRIF, TBK1, TRAF3, NEMO), (ii) HSE-causing IFN-?/?- and ??-pathway genes (STAT1 and newly discovered IFIT2), (iii) other IFN- pathway genes not related to HSE (IL10RB, TYK2, IRF7), and (iv) novel HSE-causing genes that are morbid by unknown mechanisms (DBR1, SNORA31). Second, we will devise a novel protocol to differentiate iPSCs into trigeminal neurons, where HSV-1 establishes latency in children without HSE, and compare control, TLR3- , STAT1-, IL10RB-, DBR1-, and SNORA31-mutated cells for their response to poly(I:C), IFNs, and HSV-1. Third, we will characterize the sub-cellular impact of inborn errors of TLR3, by studying the entry, retrograde axonal transport, gene delivery and replication of HSV-1 and mutants, in forebrain and trigeminal neurons from patients. To demonstrate the disease-causing role of any genetic defect, we will test isogenic iPSC lines in which the mutation has been corrected or introduced by CRISPR-Cas9 gene editing. All technologies needed have been successfully set up in a synergistic manner across our four laboratories. Exciting preliminary data have been obtained, including (i) novel genetic etiologies of childhood HSE (IFIT2, DBR1, SNORA31), (ii) novel Sendai virus (SeV)-based generation of iPSCs, (iii) novel protocols to differentiate HSV-1-permissive forebrain and trigeminal neurons, and (iv) novel imaging of HSV-1 infection in neurons. While studies of HSE have traditionally been limited to animal models, the pursuit of this human iPSC-based study will enable us to dissect in-depth the molecular and cellular basis of HSE in children with inborn errors of CNS-intrinsic immunity to HSV-1. This path-breaking collaborative study has far-reaching medical and biological implications.